1. Field of the Invention
The invention is generally related to methods of laser and/or photon transfer.
2. Description of the Prior Art
Direct write technologies have gained popularity with the increased interest in biological sensors and microarrays, and the push for engineered tissues to replace organ transplants. These techniques allow for increased ability to manipulate biological materials in very small volumes with much better accuracy than has been previously possible. Some of the most promising techniques for use in controlling and transferring biological materials are matrix assisted pulsed laser evaporation direct write (MAPLE DW, see U.S. Pat. No. 6,177,151 to Chrisey et al., incorporated herein by reference), dip-pen nanolithography (DPN), scanning probe microscopy (SPM), microcontact printing (MCP), and laser guidance direct write.
MAPLE DW focuses a pulsed laser source at the interface of a quartz “ribbon” (analogous to a typewriter, it is usually a quartz slide with a coating containing a mixture of a matrix support material and the biological materials of interest) to cause the ablation of a small amount of the interfacial matrix material, which then causes the remaining bulk matrix and biological material to be expelled from the ribbon in a bubble-jetting effect. The expelled material travels through-space, away from the laser and ribbon to a receiving substrate. This results in a spot of transferred material approximately 100 μm in diameter with pL scale volumes. MAPLE DW has limited applications or inherent limitations due to the physical properties of the biological materials and surrounding media needed to ensure accurate pattern formation. Specifically, MAPLE DW requires that a mixture of transfer material and a matrix material be presented to the laser source. The matrix must be of higher volatility than the transfer material and strongly absorb the incident radiation. In addition, the reproducibility of the technique can be low due to inconsistencies in the parameters necessary for ablation of the matrix material. Also, because the absorptivity of certain matrix materials is quite low, there is the potential for damage to biological materials from direct and indirect interaction with the incident laser radiation.
Laser transfer of biological materials presents a challenge due to the fragility of many biological materials. They can be harmed by shear stress when they are removed from the target substrate and by impact stress when they land on the receiving substrate. DNA in particular can be uncoiled by such stresses. Heat can denature many biological materials. UV damage can also result when a UV laser is used.
The analysis of tissue samples, both normal and diseased, benefits greatly from the ability to procure homogenous populations of cells directly from pathologically defined tissue sections where tremendous amounts of cellular heterogeneity can exist. This is the reasoning behind development of tissue microdissection technology as originally developed in the Laser Capture Microdissection (LCM) technology. Tissue microdissection technologies have improved the analysis of tissue samples by providing a means through which molecular profiling of cells derived from tissue samples can be placed in a pathologically relevant context. However, these technologies were originally designed for dissection of frozen tissue samples and not for use on histopathologically processed and fixed tissue samples such as formalin-fixed, paraffin-embedded (FFPE) tissue.
Other tissue microdissection technologies have been patented and tissue microdissection instruments are available on the commercial market including the PixCell systems (Arcturus), the PALM system (PALM Microlaser Technologies), the uCUT (Molecular Machines and Industries), the Leica AS LMD (Leica Microsystems), the LaserScissors (Cell Robotics), the MicroDissector (Eppendorf), and the Clonis system (Bio-Rad).
These techniques generally use one of two methods. One method is a contact method where a thin film is placed on top of and in contact with the tissue so that when a single laser event is used to illuminate through the film from the top, it activates the film to become adherent to the tissue. When the film is subsequently pulled off the tissue, the material is stuck to the underside of the film and the material is then removed from the film by biochemical procurement methods.
Another method utilizes a polyethylene, polyethylene-naphthalene, polyester, polyacrylate, polymer that contains at least 5% by weight of an aromatic or part-aromatic polycondensate coated material situated in between a planar support and the tissue. A primary laser event is used to cut around the cells of interest within the tissue and to separate these cells from the surrounding cells, and then a subsequent second laser illumination event is used to catapult those separated cells upward into a collection vessel. This method constitutes two separate laser illumination events. A third method utilizes a similar polyethylene terephthalate (PET) or polyethylene naphthalate (PEN) coating between the tissue to be dissected and the glass slide as discussed in the previous method, however, this approach relies on laser light to etch a circular boundary around the cells of interest to isolate them from the surrounding tissue after which the process of gravity causes the PET/PEN coating along with the region containing the cells of interest that has been isolated by laser etching to simply fall downwards into a collection vessel.
While each of these systems using these two methods offers unique and useful instrumentation, none were developed and optimized for formalin-fixed/paraffin embedded (FFPE) tissue as the primary tissue source for dissection. Nor have any of these previous approaches involved the transfer of tissue utilizing simple removal of the cells from the tissue by means of a single light illumination event onto a photon-absorbing material to mediate a precisely controlled explosive event for downward expulsion of the cells in a single explosive event from the tissue. The ability to perform tissue dissection from FFPE sections is not efficient with these instruments and the vast majority of tissue dissection practitioners do not utilize these instruments for that purpose. The ability to dissect cells directly from FFPE tissue sections would be advantageous.